Exploring the proton pump mechanism of cytochrome c oxidase in real time

Abstract

Cytochrome c oxidase catalyzes most of the biological oxygen consumption on Earth, a process responsible for energy supply in aerobic organisms. This remarkable membrane-bound enzyme also converts free energy from O 2 reduction to an electrochemical proton gradient by functioning as a redox-linked proton pump. Although the structures of several oxidases are known, the molecular mechanism of redox-linked proton translocation has remained elusive. Here, correlated internal electron and proton transfer reactions were tracked in real time by spectroscopic and electrometric techniques after laser-activated electron injection into the oxidized enzyme. The observed kinetics establish the long-sought reaction sequence of the proton pump mechanism and describe some of its thermodynamic properties. The 10-μs electron transfer to heme a raises the pK a of a “pump site,” which is loaded by a proton from the inside of the membrane in 150 μs. This loading increases the redox potentials of both hemes a and a 3 , which allows electron equilibration between them at the same rate. Then, in 0.8 ms, another proton is transferred from the inside to the heme a 3 /Cu B center, and the electron is transferred to Cu B . Finally, in 2.6 ms, the preloaded proton is released from the pump site to the opposite side of the membrane.